Amino terminal derivatives of RANTES are potent inhibitors of HIV-1 entry into host cells bearing the CCR5 receptor. During the first three years of this Program Project, our laboratory has analyzed both the inhibitory and agonist effects of aminooxypentane (AOP)- RANTES on the replication of primary HIV-1 isolates. This project will continue to focus on the inhibitory and stimulatory effect of PSC-RANTES, other RANTES derivatives, and fusion inhibitors on the replication of primary HIV-1 isolates. These studies will be performed in both primary blood mononuclear cells (PBMC) and Langerhans cells (LC). Preliminary results in both the Arts' laboratory using PBMC cultures and the Blauvelt's laboratory using LC in skin explants suggest that PSC-RANTES is at least 10- to 100-fold more potent than AOP-RANTES as an inhibitor of HIV-1 replication. This proposal will also assess the effects of amino acid substitutions in the env gene that have been associated with decreased sensitivity or resistance to RANTES derivatives. The laborious techniques of virus propagation and drug susceptibility testing have been improved by a method to rapidly pseudotype HIV-1 molecular clones with primary HIV-1 env genes, i.e., isolated and PCRamplified from HIV-infected individuals. Our laboratory has also been involved with several studies on HIV-1 fitness and has recently discovered a link between the relative fitness of an HIV-1 isolate and susceptibility to entry inhibitors. Specific Aim 1 will test the sensitivity of primary HIV-1 isolates to inhibition by PSC-RANTES, fusion inhibitors, and other RANTES derivatives and establish a possible correlation between susceptibility to entry inhibitors and relative fitness of RS HIV-1 isolates. In Specific Aim 2, we will determine the level of PSC-RANTES inhibition of blood- or vaginal-derived HIV-1 isolates in LC and PBMC. Dual exposure of LC to R5 and X4 HIV-1 isolates will be the assay used to establish if entry inhibitors can block primary infection by both R5 and X4 isolates. In Specific Aim 3, we will analyze the env sequences of primary isolates tested in Specific Aims 1 and 2 to identify and characterize specific amino acids associated with reduced susceptibility to PSC-RANTES inhibition. Finally, Specific Aim 4, will involve a comparison of the stimulatory and inhibitory effects of PSC-RANTES on primary HIV-1 replication. As described above, the residual agonist activity of these RANTES derivatives leads to stimulation of X4 HIV-1 replication. Finally, it is important to note that this project will be key in predicting the success of PSC-RANTES and other drugs (from Project 1) in hu-pbl-SCIDu mice (Project 3), in macaques (Project 4), and as a vaginal microbicide to block HIV-1 transmission.